Measuring G protein activation by spectrally resolved imaging fluorescence fluctuation spectroscopy

The activation of heterotrimeric G proteins through G-protein-coupled receptors (GPCRs) is a ubiquitous signaling mechanism in eukaryotic biology. The three principal molecular components of this cascade are the GPCR, Gα subunit, and Gβγ subunit. Measurement of interactions between these components and their downstream effectors in live cells is paramount to understanding how cells fine-tune their physiology in response to many external stimuli. Multicolor fluorescence fluctuation spectroscopy (FFS) approaches allow the sensitive detection of heteromeric interactions by using spectrally distinct fluorophores to label biomolecules of interest. We considered three imaging FFS approaches to measuring molecular interactions from the signals produced by a spectrally resolved confocal microscopy: raster spectral image correlation spectroscopy (RSICS), spectral spatial cumulant analysis, and native resolution spatial cumulant analysis. We characterized these approaches using simulation and experiments on heteromers with known stoichiometries. We found that RSICS had the best sensitivity for measuring heteromeric interactions and employed it to measure G protein complexes. As measured by RSICS, interactions between the G protein subunits Gαi₁ and Gβ₁γ₂ were sensitive to the stimulation of two GPCRs, the D2 dopamine receptor and the α-2A adrenergic receptor. Interactions between GPCRs and G proteins were not detectable above background, supporting a collisional model of GPCR/G protein interactions in contrast to a preassembly model where strong interactions would be present. These data are uniquely available by this FFS framework, which is appropriate for not only multiplexed measurements of G protein biology but any dynamic protein complexes in the cell.